Lethal Dengue Virus Infection Research Articles

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

Cyclovirobuxine D inhibits dengue virus replication by impeding the complete autophagy in a cholesterol-dependent manner

Dengue virus (DENV) is the most common mosquito-borne flavivirus, and it affects millions of people globally every year. Currently, there are no approved drugs for the treatment of dengue infection. By screening a natural product library, we identified a novel compound, cyclovirobuxine D (Cvb D), that displays anti-DENV activity. Cvb D inhibits DENV replication in vitro in a dose-dependent manner and protects suckling mice against lethal DENV infection. Mechanistically, Cvb D regulates the expression of genes related to the cellular cholesterol pathway. As a result, Cvb D increases cellular cholesterol synthesis and accumulation, activates mTOR, and inhibits viral-dependent autophagy. Cvb D does not suppress autophagy initiation but impedes the nuclear translocation of the lysosome transcription factor TFEB. In addition, Cvb D restricts the replication of other positive-strand RNA viruses such as Zika virus and Coxsackievirus B3. We speculate that Cvb D could be a broad-spectrum antiviral drug candidate for use against positive-strand RNA viruses that require autophagy for optimal replication.

Detrimental Role of Neutrophil Extracellular Traps during Dengue Virus Infection

A recent article by Sung etal. identified the CLEC2 platelet receptor as an important factor of lethal dengue virus infection. Formation of neutrophil extracellular traps via crosstalk with CLEC5A and TLR2 neutrophils were ascribed a causative role in DENV infection. This provides new insights for the development of candidate broad-spectrum therapies against hemorrhagic virus infections.

Increased serum sialic acid is associated with morbidity and mortality in a murine model of dengue disease.

Dengue virus (DENV) causes the most prevalent arboviral infection of humans, resulting in a spectrum of outcomes, ranging from asymptomatic infection to dengue fever to severe dengue characterized by vascular leakage and shock. Previously, we determined that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability, disrupts the endothelial glycocalyx layer (EGL) in vitro and triggers shedding of structural components, including sialic acid (Sia) and heparan sulfate. Here, using a murine model of dengue disease disease, we found high levels of Sia and NS1 circulating in mice with DENV-induced morbidity and lethal DENV infection. Further, we developed a liquid chromatography/mass spectrometry-based method for quantifying free Sia in serum and determined that the levels of free N-glycolylneuraminic acid were significantly higher in DENV-infected mice than in uninfected controls. These data provide additional evidence that DENV infection disrupts EGL components in vivo and warrant further research assessing Sia as a biomarker of severe dengue disease.

Synergism between the tyrosine kinase inhibitor sunitinib and Anti-TNF antibody protects against lethal dengue infection

Dengue virus (DENV) currently circulates in more than 100 countries and causes an estimated 390 million infections per year. While most cases manifest as a self-resolving fever, ∼1.5% of infections develop into a more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which causes ∼20,000 deaths annually. The underlying pathological feature of DHF/DSS, also known as Severe Dengue, is an acute increase in vascular permeability leading to hypovolemia and shock. Angiogenic factors and cytokines, such as vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF), have been implicated in the increased vascular permeability, suggesting a potential therapeutic strategy for Severe Dengue. Here, we employed a mouse model of antibody-dependent enhancement of DENV infection, which recapitulates the fatal capillary leakage and shock of human Severe Dengue, to investigate the effects of approved VEGF- and TNF-targeting drugs. DENV infection caused a significant increase in serum VEGF levels within 2 days and resulted in ∼80% mortality within 8 days of infection. Treatment of mice with sunitinib, a VEGF receptor tyrosine kinase inhibitor, once (day 2) or twice (days 1 and 2) post-infection reduced mortality by 50–80% compared with untreated mice. Notably, sunitinib treatment decreased serum TNF levels, white blood cell counts, and hematocrit levels relative to untreated mice, but had only marginal effects on tissue viral burden. Combination therapy with anti-TNF antibody and sunitinib significantly reduced vascular leakage and synergized to provide superior protection from lethal DENV infection compared with either agent alone. These data suggest that a two-pronged anti-angiogenic and anti-inflammatory approach may be useful for the rapid treatment of DHF/DSS.

CHARACTERISTICS OF CONTINUITY TYPE SUPPORTED BY JENTIK Aedes aegypti IN REGIONAL WORKING PUSKESMAS BULILI

Dengue Hemorrhagic Fever (DHF) is a severe and potentially lethal dengue virus infection. DHF control measures can be performed on adult mosquitoes or in larvae. Controlling the larval stage can be done by cutting the chain of transmission of dengue disease through containers that made Aedes aegypti mosquitoes laying and breeding. This study aims to determine the preferred container characteristics of larva Aedes aegypti in the work area of Puskesmas Bulili, South Palu District. This research will be carried out in the work area of Puskes Puskesmas Public Health Center by using spot survey method by observing all containers potentially become Aedes aegypti larvae breeding place in research location. The variables under study will be presented in descriptive form by describing the environmental characteristics of containers preferred by Aedes aegypti mosquito larvae ranging from type, base material and container location. The results showed that the type of container favored by Aedes aegypti mosquitoes is the type of bathtub with a base material made of plastic and the location of the container inside the community house.Keywords : DHF, Container, Aedes aegypti

Antibody Epitopes Identified in Critical Regions of Dengue Virus Nonstructural 1 Protein in Mouse Vaccination and Natural Human Infections

Dengue is a global public health problem and is caused by four dengue virus (DENV) serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV Abs can be protective or potentially increase disease via Ab-dependent enhancement. DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids Ab-dependent enhancement. In this study, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with aluminum and magnesium hydroxide, monophosphoryl lipid A + AddaVax, or Sigma adjuvant system+CpG DNA, compared with mice infected with a sublethal dose of DENV2 and mice immunized with OVA (negative control). We characterized Ab responses to DENV1, 2, and 3 NS1 using an Ag microarray tiled with 20-mer peptides overlapping by 15 aa and identified five regions of DENV NS1 with significant levels of Ab reactivity in the NS1 + monophosphoryl lipid A + AddaVax group. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent, and 12-mo timepoints. One region in the wing domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

A nuclear transport inhibitor that modulates the unfolded protein response and provides in vivo protection against lethal dengue virus infection.

Dengue virus (DENV) is estimated to cause 390 million infections each year, but there is no licensed vaccine or therapeutic currently available. We describe a novel, high-throughput screen to identify compounds inhibiting the interaction between DENV nonstructural protein 5 and host nuclear transport proteins. We document the antiviral properties of a lead compound against all 4 serotypes of DENV, antibody-dependent enhanced (ADE) infection, and ex vivo and in vivo DENV infections. In addition, we use quantitative reverse-transcription polymerase chain reaction to examine cellular effects upon compound addition. We identify N-(4-hydroxyphenyl) retinamide (4-HPR) as effective in protecting against DENV-1-4 and DENV-1 ADE infections, with 50% effective concentrations in the low micromolar range. 4-HPR but not the closely related N-(4-methoxyphenyl) retinamide (4-MPR) could reduce viral RNA levels and titers when applied to an established infection. 4-HPR but not 4-MPR was found to specifically upregulate the protein kinase R-like endoplasmic reticulum kinase arm of the unfolded protein response. Strikingly, 4-HPR but not 4-MPR restricted infection in peripheral blood mononuclear cells and in a lethal ADE-infection mouse model. 4-HPR is a novel antiviral that modulates the unfolded protein response, effective against DENV1-4 at concentrations achievable in the plasma in a clinical setting, and provides protection in a lethal mouse model.

Correction: Therapeutic Efficacy of Antibodies Lacking FcγR against Lethal Dengue Virus Infection Is Due to Neutralizing Potency and Blocking of Enhancing Antibodies

There is an error in the article title, which is missing the word binding. The title should instead read: Therapeutic Efficacy of Lacking Fcγ Receptor Binding against Lethal Dengue Virus Infection Is Due to Neutralizing Potency and Blocking of Enhancing Antibodies

Therapeutic Efficacy of Antibodies Lacking FcγR against Lethal Dengue Virus Infection Is Due to Neutralizing Potency and Blocking of Enhancing Antibodies

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV). At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs) and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.

Competitive inhibitor of cellular α-glucosidases protects mice from lethal dengue virus infection

Dengue virus infection causes diseases in people, ranging from the acute febrile illness dengue fever, to life-threatening dengue hemorrhagic fever/dengue shock syndrome. We previously reported that a host cellular α-glucosidases I and II inhibitor, imino sugar CM-10–18, potently inhibited dengue virus replication in cultured cells, and significantly reduced viremia in dengue virus infected AG129 mice. In this report we show that CM-10–18 also significantly protects mice from death and/or disease progress in two mouse models of lethal dengue virus infection. Our results thus provide a strong support for the development of CM-10–18 or its derivatives as antiviral agents to treat servere dengue virus infections.

The Human Immune Response to Dengue Virus Is Dominated by Highly Cross-Reactive Antibodies Endowed with Neutralizing and Enhancing Activity

Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcγ receptors (FcγR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII- or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcγR binding did not enhance infection and neutralized DENV invitro and invivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibody-based therapy to control severe dengue.

Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in Escherichia coli are protected against lethal dengue virus infection

A gene fragment encoding the C-terminal 204 amino acids (AA) from the structural envelope glycoprotein (E) and the N-terminal 65 AA from non-structural protein one (NS1) of dengue type 2 virus (DEN-2) was expressed in Escherichia coli (E. coli) as a fusion protein with staphylococcal protein A. The recombinant fusion protein was purified and analysed for its antigenicity, its immunogenicity and its ability to protect mice against lethal challenge with live DEN-2 virus. The recombinant protein was found to be reactive with anti-DEN-2 polyclonal and monoclonal antibodies. Mice immunized with the purified fusion protein made anti-DEN-2 antibodies measured by the hemagglutination-inhibition (HI) and neutralization (N) tests, and were protected against lethal challenge with DEN-2 virus administered by intracranial inoculation.

Passive protection studies in mice with monoclonal antibodies directed against the non-structural protein NS3 of dengue 1 virus

Antibody-mediated enhancement of dengue virus replication is thought to be a mechanism contributing to the pathogenesis of dengue haemorrhagic fever and dengue shock syndrome. Enhancement is associated with antibodies to structural components of the virus. To circumvent the problem of immune enhancement, studies to identify protective antigens of dengue virus have involved non-structural proteins. Passive and active protection against lethal dengue virus infection in mice have been demonstrated with the non-structural protein NS1. In this study, the dengue virus non-structural protein NS3 was examined in passive protection studies with monoclonal antibodies prepared against NS3 of dengue 1 virus (Hawaiian). Five monoclonal antibodies that were authenticated to be reactive to NS3 were used to immunize 13- to 14-day old mice intraperitoneally. Thereafter, the mice were challenged intracerebrally with 100 LD50 of neurotropic dengue 1 virus and the survival indices of the mice were calculated. Significant decreases in survival indices (P less than 0.05), indicating increases in survival times were observed with four of five monoclonal antibodies tested. Monoclonal antibodies to NS3 of dengue 1 virus are able to increase the survival time of mice challenged with a lethal dose of dengue 1 virus, although the mechanism remains to be defined.

Failure of a dengue 1 sub-unit vaccine to protect mice against a lethal dengue virus infection.

Mice immunized with DEN-1 pre-matrix protein produced antibody which reacted with dengue virus infected cells and intact virions but failed to neutralize virus in vitro or in vivo. Newborn mice passively immunized with anti-DEN-1 pre-matrix antibody also failed to survive intracerebral infection with 100 LD50 of any dengue virus serotype.